登革病毒2型广东省分离株的鉴定及NS1基因序列分析

目的 明确广东省南海市1998年夏、秋季一批发热、出疹患者的诊断,并从基因水平分析其流行株的来源。方法 收集疑似登革热（DF）患者 血清52份,应用逆转录－聚合酶链反应（RT－PCR）、病毒分离和间接免疫荧光技术分别进行了病原学和血清学检测;同时采用分子克隆技术将PCR扩增产物克隆到T载体,并进行序列测定。结果 从25份发病早期患者血标本中分离病毒10份,经用单克隆抗体间接免疫荧光检测和RT－PCR检没让实为登革病素2型感染。基因序列分析表明,1998年广东分离的登革2型病毒与ThNH-P28/93、C0166`16681`NGC`JAM`04`GD06/93和S1株核苷酸的同源性分别是：98％、97％、96％、93％、92％、91％。结论 此次南海市登革热暴发流行为 登革病毒2型感染所致。基因序列分析提示：1998年广东省南海市分离的登革病毒2型可能来源于泰国。     

云南登革4型病毒的鉴定及NS1和NS2a基因序列分析

目的对1989年8月分离自云南省盈江县蚊虫的2株登革病毒进行生物学及分子特征的鉴定,明确这两株病毒的血清型及基因型。方法蚊虫用BHK21细胞和乳鼠法分离病毒,用间接免疫荧光试验、RT-PCR等方法进行鉴定,并对这两株病毒的分子生物学特征进行分析。结果从白纹伊蚊和达勒姆阿蚊中各分离到一株病毒,分别命名为M110和M113,这两株病毒对BHK21细胞能产生明显的细胞病变,脑内接种乳鼠4d左右导致乳鼠死亡。该病毒经血凝、血凝抑制和间接免疫荧光试验提示为黄病毒。分别用黄病毒属特异引物、登革4型病毒NS1和NS2a基因片段特异引物进行RT-PCR扩增、序列测定,证实为登革4型病毒。NS1和NS2a基因序列片段分析表明,M110和M113的NS1核苷酸序列与登革4型病毒基因Ⅰ型的H241株(Y19176)同源性最高,分别为99%、98%;NS2a基因片段与H241的核苷酸序列的同源性分别为96.5%、97.1%。结论从盈江县蚊虫分离到的M110和M113病毒为登革4型病毒基因Ⅰ型,表明云南省西部边境地区在20世纪80年代发生过登革4型病毒的流行。     

Epidemiological and entomological surveillance of the co-circulation of DEN-1, DEN-2 and DEN-4 viruses in French Guiana

We surveyed the disease epidemiology of dengue in French Guiana after the first dengue haemorrhagic fever epidemic from 1991 to 1993 and during an endemic period from 1993 to 1995. DEN-1, DEN-2 and DEN-4 viruses were isolated from patients and DEN-4 was also isolated from Aedes aegypti mosquitoes. Cases of dengue were reported from all over the country, not only from urban areas, but also from rural areas and isolated human settlements, indicating widespread circulation of the viruses. The mosquito vector A. aegypti was found in all inhabited areas of French Guiana and small outdoor containers were the most common breeding grounds. Some ecological features of A. aegypti, such as larvae breeding in Bromeliad plants in the rainforest, a non-exclusive anthropophily and a high vertical transmission rate for dengue viruses, indicate that A. aegypti can behave as a reservoir for dengue viruses in silent areas. Dengue viruses may survive at an endemic level and cause outbreaks when unknown conditions become more favourable. This finding adds to our knowledge of the natural history of dengue viruses in the Americas.     

H5N1亚型和H3N2亚型禽流感病毒广西株NS基因的克隆和序列分析

目的对3株分离自广西的禽流感病毒的NS基因进行序列分析,试图从分子水平分析和了解广西地区禽流感病毒NS基因的变异特点和进化规律。方法根据GenBank上登录的已知禽流感病毒的NS基因全序列设计引物,对3株2004-2005年分离自广西的禽流感病毒株A/DK/GX/1566/04(H3N2)、A/Goose/GX/737/05(H5N1)、A/Goose/GX/2775/05(H5N1)的cDNA进行PCR扩增NS基因,并将其克隆到pMD-18T载体上,分别获得全长为855bp、834bp、837bp的NS基因全序列。结果2株H5N1亚型禽流感病毒广西株间NS基因序列的同源性为95.3%,而2株H5N1亚型禽流感病毒株与H3N2亚型禽流感病毒株之间NS基因序列的同源性为94.0%～94.1%。H5N1亚型禽流感病毒广西株的NS基因在第238-253位核苷酸处均发生了15个核苷酸缺失,与我国广东、香港、云南、湖南、湖北及东南亚等不同地区的毒株比较,NS基因的核苷酸同源性为70.2%～97.6%。在NS基因系统进化树中,3株禽流感病毒广西株都属于A亚群,但处在不同的分支上,其中A/DK/GX/1566/04(H3N2)与广东、香港2001-2003年分离株处于同一分支;A/Goose/GX/737/05(H5N1)、A/Goose/GX/2775/05(H5N1)与我国华南地区以及韩国、越南、泰国等亚洲东南部国家的2003年以后的分离株有共同起源。结论3株2004-2005年分离自广西的禽流感病毒株NS基因之间的同源性均高于94%,都属于A亚群。     

两株B型流感病毒的基因组序列分析

目的 了解两株B型流感病毒的基因组序列特征。方法 对两株B型流感病毒进行全基因组序列测定和基因进化特性的分析。结果 成功扩增出两株B型流感病毒的全基因组序列,并且两毒株的基因组均属于Yamagata系类似株Ⅱ系。两株病毒的基因组序列与疫苗株B/Florida/4/2006(Yamagata系)以及Victoria系代表株B/Brisbane/60/2008、B/Malaysia/2506/2004相比,HA基因同源性为89.7%～97.3%,NB基因同源性则为95.4%～98.4%。两株病毒的HA蛋白与WHO推荐的疫苗株B/Florida/4/2006(Yamagata系)相比,存在11处氨基酸突变,其中HA的N131K氨基酸位点突变,可能对抗原性产生影响。NB的S198N氨基酸位点突变可能影响神经氨酸酶抑制剂的灵敏度。结论 两株B型流感病毒的遗传进化状态稳定,但是与同分支中的毒株基因序列部分位点存在差异。     

猪Torque teno病毒广东株的PCR检测和序列分析

Torque teno病毒（Torque teno virus,TTV）是近年来才发现的一种单股负链DNA病毒。猪Torque teno病毒（Torque teno sus virus, TTsuV）在猪群中大量流行,被认为与猪的断奶后多系统衰竭综合征(post-weaning multi-systemic wasting syndrome, PMWS)等疾病有关。本文对来自广东的2个猪场的14份猪血清进行了PCR检测,扩增出猪Torque teno病毒的UTR片段,共检出10份TTsuV1阳性（71%）,8份TTsuV2阳性（57%）,其中TTsuV1和TTsuV2双重阳性的为5份（36%）。PCR产物与参考毒株相应序列的分析证实,GDT1-1和GDT1-2的UTR片段均与TTsuV1参考毒株高度相似, GDT2-1和GDT2-2的UTR片段与TTsuV2参考毒株高度相似。     

Nucleotide sequence of influenza virus RNA segment 8 indicates that coding regions for NS1 and NS2 proteins overlap.

The smallest RNA segment of influenza A viruses (vRNA segment 8) has recently been shown to code for two unrelated nonstructural proteins (NS1 and NS2) translated from separate mRNAs. Molecular weight considerations indicated that there might not be enough space on vRNA segment 8 for the two coding regions unless they overlap. We have recently cloned in bacterial plasmids several genes of an avian influenza A virus, fowl plague virus (EPV), and now present the complete nucleotide sequence of FPV RNA segment 8 largely determined from the cloned DNA. The DNA sequence predicts two open protein synthesis reading frames that can be translated into polypeptides of sizes similar to those of NS1 and NS2. The coding regions for these polypeptides overlap by the equivalent of 43-60 amino acids, the exact amount depending on which of several possible methionines initiates the synthesis of NS2.     

4株狂犬病街毒的分离及其核蛋白和糖蛋白序列分析

目的以4株狂犬病街毒分离株及我国南北方不同地区狂犬病病毒街毒为研究对象,通过其核蛋白及糖蛋白基因的同源性及进化分析,比较我国狂犬病流行毒株与人用及兽用狂犬病疫苗株间的基因及抗原性差异,为相关疫苗的研究奠定理论基础。方法以直接免疫荧光法(DFA)检测疑似狂犬病的犬脑标本,以乳鼠颅内接种法(MIT)和细胞培养分离技术(CIT)分离狂犬病病毒,并进行TCID50和LD50测定;以RT-PCR法扩增其核蛋白(N)和糖蛋白(G)基因,克隆入T载体并测序后,下载GenBank内已有的狂犬病病毒数据资源,以(Clustal和MEGA3软件)进行基因同源性比对及系统发生分析。结果从广东、河北两省分离到狂犬病病毒街毒4株,分别命名为GN07、WJ07-1、WJ07-2、GC07;序列分析表明4株狂犬病病毒均为基因1型,其N基因与G基因核苷酸的同源性分别为89.5%~99.9%和87.9%~99.9%,其中GN07与WJ07-1、WJ07-2、GC07的同源性较低。与己知的基因1型狂犬病毒相比,WJ07-1、WJ07-2、GC07与湖南、湖北、云南、浙江等地分离株及印度尼西亚分离株核苷酸同源性最高,分别为97.8%~99.4%和92.2%;GN07与CTN疫苗株核苷酸同源性最高,为94.5%。系统发育分析表明,4株病毒与CTN疫苗株同源性较高,与人用狂犬病疫苗3aG、PV株及兽用狂犬病疫苗ERA株和Flury疫苗株的同源性相对较远。适应细胞培养后,四株街毒的LD50和TCID50最高者为GN07,而WJ07-1较低,差别较大。结论4株狂犬病分离株与我国南北方不同地区狂犬病流行毒株的基因和氨基酸序列存在一定差异,在进化关系上分属于不同的分支。     

Interferon-beta is activated by hepatitis C virus NS5B and inhibited by NS4A, NS4B, and NS5A

Innate immunity is part of the antiviral response. Interferon (IFN)-beta plays a leading role in this system. To investigate the influence of hepatitis C virus (HCV) on innate immunity, we examined the effect of viral proteins on IFN-beta induction. HepG2 cells were co-transfected with plasmids for seven HCV proteins (core protein, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) and the IFN-beta promoter luciferase. Toll-like receptor (TLR) 3 and Toll/IL-1 receptor domain-containing adapter inducing IFN-beta (TRIF) play key roles in dsRNA-mediated activation of interferon regulatory factor (IRF)-3 and IFN-beta; therefore, the participation of TLR3/TRIF in NS5B-mediated IFN induction was examined. Among seven HCV proteins, only NS5B, a viral RNA-dependent RNA polymerase (RdRp), activated the IFN-beta promoter. However, mutant NS5B without RdRp activity or template/primer association did not activate the IFN-beta promoter. Activation of the IFN-beta promoter by NS5B required the positive regulatory domain III, a binding sequence for IRF-3. Moreover, IRF-3 was phosphorylated by NS5B. Both inhibition of TLR3 expression by small interfering RNA and expression of the dominant negative form of TRIF significantly reduced NS5B-induced activation of IFN-beta. Of the six other HCV proteins, NS4A, NS4B, and NS5A efficiently inhibited this activation. HCV NS5B is a potent activator of the host innate immune system, possibly through TLR3/TRIF and synthesis of dsRNA. Meanwhile, NS4A, NS4B, and NS5A block IFN-beta induction by NS5B, which may contribute toward the persistence of this virus.     

粪便中戊型肝炎病毒散发株的核酸序列分析

目的了解粪便中戊型肝炎病毒（ＨＥＶ）散发株的核酸序列变异情况。方法用逆转录－套式－聚合酶链反应（ＲＴ－Ｎｅｓｔｅｄ－ＰＣＲ）检测了８例广州地区散发性戊型肝炎患者粪便中的ＨＥＶＲＮＡ，３例阳性。对阳性ＰＣＲ产物的２３８个碱基进行了基因克隆和核酸序列分析，并与已报道的ＨＥＶ序列进行比较。结果本地区３株ＨＥＶ与缅甸株（Ｂ）、巴基斯坦株（Ｐ）、墨西哥株（Ｍ）、中国新疆株（Ｃｈ１．１）和广州血清株（Ｇ－９）的核苷酸和氨基酸的同源性均值分别为８０．６７％和８８．６０（Ｂ）、８１．２５％和８９．２０％（Ｐ）、７７．４５％和８４．８１％（Ｍ）、８１．２５％和８９．２０％（Ｃ）、９７．８５％和９６．２０％（Ｇ）。结论本组３株ＨＥＶ有一定程度的变异。     

山东省丙型肝炎病毒分离株NS5区核苷酸序列分析

目的：了解山东省丙型肝炎病毒（HCV）分离株的基因型及其基因的变异情况,方法：应用德国UBI HCV EIA 4．0诊断试剂盒筛选山东省部分地区64例临床检验为抗－HCV阳性的血清标本,有54例阳性,随意抽取其中28例,应用逆转录套式－聚合酶链反应（RT－nested-PCR)扩增319bp的HCV NS5区基因片段,结果12例出现特异性条带,随后将这12个NS5区片段直接进行T载体克隆,并用Sanger法对克隆成功的10个NS5区基因片段进行序列测定,将所得到的10个序列与GenBank中所有的HCV分离株进行同源性比较。结果：10例山东省部分地区HCV分离株的基因型均属于HCV－1b型,对获得的10个NS5区片段进行是性分析发现,所有的核苷酸变化都是由于替代作用引起的,没有碱基的插入和缺失;大部分的突变属于同义突变,占突变总数的74％。RNA－依赖性RNA聚合酶的G－D－D基序和所有的半胱氨酸都完全保守,结论：本研究证明了HCV－1b型是山东省部分地区主要的基因型。     

Cross-genotypic interaction between hepatitis C virus NS3 protease domains and NS4A cofactors

BACKGROUND/AIMS: Hepatitis C virus (HCV) nonstructural protein 3 (NS3) protease requires NS4A as a cofactor. This cofactor activity has been mapped to the central region of NS4A which interacts with the N-terminus of NS3 protease. To investigate whether this interaction is conserved among different genotypes of HCV cross-genotypic characterization were performed to delineate the importance of NS4A cofactor function in relation to the molecular evolution of HCV METHODS: Active NS3 protease domains of genotype 1-3 (representing five subtypes: la, 1b, 2a, 2b and 3a) were produced and purified from bacterial cells. NS4A cofactor-dependent in vitro trans cleavage assays were established using the in vitro translated recombinant protein substrates. These substrates contained the junction site of NS4A/NS4B, NS4B/NS5A or NS5A/NS5B. RESULTS: Our data revealed that NS3 proteases cross-interacted with NS4A cofactors derived from different genotypes, although the genotype 2 cofactor was less efficient, which could be due to greater genetic variations in this region. Furthermore, the corresponding region in hepatitis G virus (HGV) NS4A was found to provide weak cofactor activity for HCV NS3 protease. Surprisingly, a synthetic substrate peptide from the NS4B/NS5A junction was also found to enhance HCV NS3 protease activity in a dose-dependent manner. CONCLUSION: Our study suggests that the NS4A cofactor function is well conserved among HCV It is likely that other HCV-related viruses may have developed similar strategies to regulate their protease activity.     

中国地区HCV株包膜区cDNA序列变异性分析

目的　研究国内不同地区ＨＣＶ包膜基因区变异。方法　对国内不同地区慢性丙型肝炎患者的１３株 ＨＣＶ采用ＲＴ－ＰＣＲ技术扩增ＨＣＶ包膜基因区（Ｅ１、 Ｅ２／ＮＳ１）片段，对其中的１００５ｂｐ的ｃＤＮＡ进行测序，并与 国内外株进行变异性分析。结果　国内ＨＣＶ株比较，同型（Ⅱ）间该区核苷酸和氨基酸的同源性分别介于７６．４２％～ ９７．１１％和７５．２２％～９３．１５％，不同型（Ⅲ）间分别为６２．２９％～６３．３８％和５１，２９％～６０．００％；国内与国际同型株比较，同型 （Ⅱ）间分别为７３．７３％～８７．７６％和７５．８２％～８８．０９５％，不同型（Ⅰ、Ⅱ、Ⅲ）间分别为６１．４９％～８１．４９％和４９．３５％～７８．２１％；国 内外１８株ＨＣＶ的ＨＶＲ１区同源性仅为３６．１１％～７５、３１％和２０．８０％～６２．９６％，发现两个新的较高变异区（第２５０～２５７ 位和第４４３～４６５位氨基酸）。结论　ＨＣＶ包膜区序列分析同型间同源性较高，异型间同源性较低；南北地区有差 异，国内外差异更大；最大的变异在ＨＶＲ１，并发现两个新的较高变异区。     

Complete nucleotide sequence of an Indian strain of Japanese encephalitis virus: sequence comparison with other strains and phylogenetic analysis.

The RNA genome of an Indian strain of Japanese encephalitis virus (JEV), GP78, was reverse transcribed and the cDNA fragments were cloned in bacterial plasmids. Nucleotide sequencing of the cDNA clones covering the entire genome of the virus established that the GP78 genome was 10,976 nucleotides long. An open reading frame of 10,296 bases, capable of coding for a 3,432 amino acid polyprotein, was flanked by 95- and 585-base long 5'- and 3'-non-coding regions, respectively. When compared with the nucleotide sequence of the JaOArS982 strain, the JEV GP78 genome had a number of nucleotide substitutions that were scattered throughout the genome except for the 5'-noncoding region, the sequence of which was fully conserved. Comparison of the complete genome sequences of different JEV isolates showed a 1.3-4.1% nucleotide sequence divergence among them, which resulted in 0.6-1.8% amino acid sequence divergence. Analysis based on the complete genome sequences of different JEV isolates showed that the GP78 isolate from India was phylogenetically closer to the Chinese SA14 isolate.     

丙型肝炎病毒非结构蛋白4B对肝癌细胞增殖的影响

目的 探讨丙型肝炎病毒非结构蛋白4B(HCV-NS4B)对肝癌细胞增殖及增殖细胞核抗原(PCNA)和周期素D1(cyclinD1)蛋白表达的影响.方法 采用构建好的HCV-NS4B表达载体,以脂质体转染法转染HepG2细胞.RT-PCR、琼脂糖凝胶电泳证实HCV-NS4B在HepG2细胞稳定表达.MTT法绘制生长曲线,观察NS4B对肝细胞生长的影响;流式细胞仪检测细胞周期的情况;免疫细胞化学法检测PCNA和cyclinD1的表达.结果 转染后,生长曲线显示NS4B可促进肝细胞生长;细胞周期检测显示转染NS4B的HepG2细胞进入S期和G2/M期增多(P＜0.05),处于G0/G1期细胞降低(P＜0.05).PCNA和cyclinD1的表达均较空白载体组升高(P＜0.05).结论 HCV-NS4B可干扰肝癌细胞周期,促进肝癌细胞增殖,其作用与上调PCNA、cyclinD1表达有关.     

Expression and epitope analysis of hepatitis C virus NS1

A cDNA encoding putative NS1 of hepatitis C virus (HCV) was expressed inEscherichia coli. The recombinant product reacted with antibodies in sera from patients with hepatitis C. For epitope analysis, immunoreactivities of synthetic peptides deduced from the NS1 region were examined. Three peptides corresponding to amino acid residues 384–411, 514–550 and 639–667 reacted with antibodies in sera from patients with hepatitis C.     

Point mutations in E2, NS3 and NS5A of hepatitis G virus

AIM To compare the point mutation deviations of HGV among E2, NS3 and NSSA.METHODS Seven patients with hepatic diseases from Japan and China were selected for this study. RNAwas extracted and amplified by semi-nested RT-PCR; and the PCR products were sequenced directly.RESULTS The point mutation deviations of HGV ia E2, NS3 and NS5A were 10% - 17%, 11% -23%,and 0% - 5%, in nuclcotide sequences and 4% - 12%, 0%, and 0% - 6% in amino acid sequencesrespectively.CONCLUSION The frequency of variation at the nucleotide level was in the order NS3>E2>NS5A, whileat the amino acid level the order was E2 >NS5A>NS3. The detected sequences from the N-terminus of E2may be the poorly conserved region of HGV.     

丙型肝炎病毒NS5A在肝癌细胞对TLR4表达的影响

目的探讨丙型肝炎病毒非结构蛋白质5A（HCV NS5A）对Toll样受体4（TLR4）表达的影响。方法用含HCV NS5A基因的表达质粒pcNS5A瞬时转染QSG7701细胞,采用免疫细胞化学技术检测HCV NS5A及TLR4蛋白质的表达;采用逆转录聚合酶链反应观察HCV NS5A和TLR4的mRNA表达。结果转染pcNS5A质粒细胞内有HCV NS5A蛋白的表达。转染pcNS5A组TLR4 mRNA及蛋白质表达均较转染pRc/CMV和未转染质粒组明显增强,而后两组细胞TLR4 mRNA和蛋白质表达相似。结论 HCV NS5A可上调TLR4 mRNA和蛋白的表达。     

Conserved nucleotide sequence of NS5A in hepatitis G virus or GB virus C in Japan and China

A semi-nested PCR method was designed for detecting the NS5A region of hepatitis G virus (HGV) or GB virus C (GBV-C) using specific oligonucleotide primers. Patients, 14 of 69, suffering from hepatic diseases in Japan and 5 of 21 in China showed strong positive signals for HGV or GBV-C by this method. The PCR products were directly sequenced. The nucleotide sequences identified differed from one another by 0–5%, from HGV by 3–9% and GBV-C by 2–7%, the percentages of the latter two being compared with the sequences reported previously. Amino acid sequences were the same or had only one alteration compared with HGV and two to three alterations compared with GBV-C. These results indicate that the nucleotide sequence of NS5A of HGV (or GBV-C) is more conserved than that of NS3 of GBV-C (or HGV) which has been reported to show variations of 0–23%.